Date of Award

12-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Plant and Environmental Science

Committee Chair/Advisor

Dr. Sachin Rustgi

Committee Member

Dr. Daniel J. Anco

Committee Member

Dr. Hong Luo

Committee Member

Dr. Sourabh Dhingra

Abstract

Peanut allergy, a critical health concern with high morbidity and mortality rates, is a leading cause of fatal anaphylactic reactions in the United States. Managing peanut allergy is challenging due to the prevalence of peanuts in processed foods and the risk of contamination. This dissertation evaluates the U.S. peanut mini-core collection for four major immunogenic proteins, Ara h1, Ara h2, Ara h3, and Ara h6, using gel electrophoresis, enzyme-linked immunosorbent assay, liquid chromatography, and mass spectrometry. The study identified 26 genotypes with reduced concentrations of allergenic proteins. Genome-wide association studies (GWAS) of protein traits revealed 10 significant SNPs and identified candidate genes that enhanced our understanding of the genetic regulation of immunogenic peanut proteins. In light of the increasing prevalence of peanut allergy and the challenges of breeding for reduced allergen content using conventional methods, this research also explored multigene editing using protoplast transformation and viral-mediated as well as biolistic delivery for stable transformation and site-directed mutagenesis. The CRISPR/Cas12a (Cpf1) system, a less-explored yet powerful tool in crop improvement, was applied to tetraploid peanut protoplasts, targeting Ara h1, Ara h2, Ara h3, and Ara h6, achieving a 56.49% efficacy. The induced mutations, included 5.01% insertions and 31.04% deletions. The tomato yellow leaf curl virus-mediated delivery of CRISPR/Cas12a further demonstrated effective mutagenesis, highlighting the potential to using viruses to deliver the genome editing reagents to plants. Additionally, the genome editing constructs, pTA001 and pTA002, were delivered using a biolistic approach, confirmed by GFP markers. Next-generation and Sanger sequencing identified a large number of mutations in 23 T0 plants. Specifically, significant mutation patterns were observed in the Ara h1 gene, with monoallelic, biallelic, and triallelic mutations at rates of 1.5%, 10.93%, and 23.43%, respectively. The Ara h 2 and Ara h 3genes also exhibited substantial biallelic and triallelic mutations, indicating the efficacy of CRISPR/Cas12a in generating multigenic edits and its potential in developing peanut genotypes with reduced content of allergenic proteins. The dissertation also addresses aflatoxin contamination by Aspergillus flavus using RNA interference (RNAi) with a non-transgenic approach. A non-pathogenic E. coli strain HT115(DE3) produced and delivered dsRNAs targeting the AflC and Cyp51 genes. AlfC-HT115 suppressed AflC gene expression and aflatoxin production, while Cyp51-HT115 suppressed A. flavus growth on peanut cotyledons. These findings underscore the potential of dsRNA-based interventions for aflatoxin management and the effectiveness of bacterial delivery system.

Author ORCID Identifier

https://orcid.org/0000-0002-7147-1542

Available for download on Wednesday, December 31, 2025

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